Rhodopsin TM6 Can Interact with Two Separate and Distinct Sites on Arrestin: Evidence for Structural Plasticity and Multiple Docking Modes in Arrestin–Rhodopsin Binding

Abstract

Various studies have implicated the concave surface of arrestin in the binding of the cytosolic surface of rhodopsin. However, specific sites of contact between the two proteins have not previously been defined in detail. Here, we report that arrestin shares part of the same binding site on rhodopsin as does the transducin Gα subunit C-terminal tail, suggesting binding of both proteins to rhodopsin may share some similar underlying mechanisms. We also identify two areas of contact between the proteins near this region. Both sites lie in the arrestin N-domain, one in the so-called “finger” loop (residues 67–79) and the other in the 160 loop (residues 155–165). We mapped these sites using a novel tryptophan-induced quenching method, in which we introduced Trp residues into arrestin and measured their ability to quench the fluorescence of bimane probes attached to cysteine residues on TM6 of rhodopsin (T242C and T243C). The involvement of finger loop binding to rhodopsin was expected, but the evidence of the arrestin 160 loop contacting rhodopsin was not. Remarkably, our data indicate one site on rhodopsin can interact with multiple structurally separate sites on arrestin that are almost 30 Å apart. Although this observation at first seems paradoxical, in fact, it provides strong support for recent hypotheses that structural plasticity and conformational changes are involved in the arrestin–rhodopsin binding interface and that the two proteins may be able to interact through multiple docking modes, with arrestin binding to both monomeric and dimeric rhodopsin.

Conformational Selection and Equilibrium Governs the Ability of Retinals to Bind Opsin

“…As depicted in Fig. 4A, the Arr peptide binds in the same cleft in MII as the G t C-term peptide, shown here as peptide fragments bound in opsin crystal structures (4,17,(53)(54)(55)(56). Interestingly, we found that the Arr peptide increased the rate and amount of ATR binding to M257Y-CAM (Fig.…”

“…Shifting the ops 3 ops* equilibrium by binding of peptide mimetics G t C-term peptide (green) and Arr peptide (purple) enhances the rate of ATR binding, and G t C-term peptide also slows 11CR binding. A, schematics depicting the full-length G protein and arrestin and how the G t C-term and Arr peptide bind in the same cytoplasmic cleft of rhodopsin (3,4). The regions of the full proteins corresponding to the peptides are highlighted in green on the G protein and purple on the arrestin.…”

Functional competence of a partially engaged GPCR–β-arrestin complex

“…Therefore, we first designed a cysteine-less βarr1 mutant and then exchanged Leu 68 in the finger loop with a cysteine (referred to as βarr L68C ). We selected L 68 C based on previous studies with rhodopsin-visual arrestin system that have used the corresponding position (L 72 C) in the finger loop56192022. We subsequently purified βarr1 L68C and labelled it with an environmentally sensitive fluorophore monobromobimane (mBBr) at Cys 68 .…”

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

“…Binding of either Gt or arrestin can cause incomplete ATR release from photoactivated rhodopsin (12,25). The presumption has been that the retinal release is blocked, causing the ATR to be "trapped" inside the receptor binding pocket (12,13). However, our results with the CAM, M257Y, made us rethink what actually causes retinal "trapping."…”

“…Fluorescence spectroscopy was carried out as previously described using a modified Photon Technology International steady-state fluorometer with the excitation source replaced with OceanOptics LEDs, LLS-295 and LLS-405 (8,13). A bifurcated fiber optic cable allowed for dual excitation of both intrinsic tryptophans and a bimane probe.…”